The present invention relates to a process for the enzymatic preparation of protected di- or oligopeptides in highly concentrated, aqueous solutions.
Synthetic short-chain peptides are increasingly being used in pharmacology and parenteral nutrition. Exemplary of such pharmacologically active dipeptides is kyotorphin (L-Tyr-L-Arg) which promotes the release of enkephalins. Enkephalins are endogenous substances which exhibit an analgesic and sedative effect in the brain (Hughes, 1975).
The chemical and optical purity of peptides intended for such a use are very critical. Accordingly, it is increasingly common to employ regio- and stereospecific enzymatic peptide syntheses to produce such peptides. Such syntheses usually entail reacting, under kinetic control, N-protected amino acid alkyl esters in the presence of a hydrolase with an amino acid derivative or a di- or oligopeptide derivative with a free amino group. When such reactions are carried out in the presence of water, the alkyl esters will also undergo hydrolysis as this reaction is thermodynamically favored.
A disadvantage associated with this process is that the enzymes employed in these syntheses are only active and stable in the presence of water, while the substrates are generally only slightly soluble in water. Adding organic solvents in order to increase the solubility of the substrates usually at least partially inactivates an enzyme and causes a decrease in its stability. As a result, prior to the instant invention, the maximum substrate concentration employed has usually been only 100 mM. In addition, prior to the instant invention, nucleophilic components have been employed in large excesses (2 to 20-fold) in order to increase the selectivity of the reaction and thereby improve the yield. In chemical syntheses of short-chain peptides in liquid phase, the substrate and nucleophile are normally employed in high and in virtually stoichiometric concentrations. Employing the substrate and nucleophile in such concentrations has not hitherto been possible for the regio- and stereospecific enzymatic peptide syntheses of the present invention.